Download Lysis-deficient phages as novel therapeutic agents for controlling

Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
RESEARCH ARTICLE
Open Access
Lysis-deficient phages as novel therapeutic
agents for controlling bacterial infection
Vivek Daniel Paul1,2, Sudarson Sundarrajan1, Sanjeev Saravanan Rajagopalan1, Sukumar Hariharan1,
Nanjundappa Kempashanaiah1,3, Sriram Padmanabhan1,4, Bharathi Sriram1* and Janakiraman Ramachandran1
Abstract
Background: Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic
resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns
over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose
determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would
alleviate these concerns without compromising efficacy.
Results: We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin
gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S.
aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted
by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid
construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol.
Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by
cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques
only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the
parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised
mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the
endolysin-deficient phage rescued mice from the fatal S. aureus infection.
Conclusions: A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to
methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode
utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage
effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient
staphylococcal phage.
Background
Bacteriophages are attractive as therapeutic agents because
they are safe for humans and highly specific and lethal to
the bacteria they target. Further, phages can be developed
rapidly to combat the emergence of antibiotic-resistant
pathogenic bacteria [1,2]. Phage therapy is currently practiced routinely and successfully in countries such as
Poland and Russia [3]. The recent approval of commercial
phage preparations by the United States Food and Drug
Administration to prevent bacterial contamination of meat
* Correspondence: [email protected]
1
Gangagen Biotechnologies Pvt Ltd, No. 12, 5th Cross, Raghavendra Layout,
Tumkur Road, Yeshwantpur, Bangalore-560 022, India
Full list of author information is available at the end of the article
and poultry [4] may pave the way for the global use of
phage therapy to control bacteria in human infections.
The development of phages for therapy has been hampered by concerns over the potential for immune
response, rapid toxin release by the lytic action of phages,
and difficulty of dose determination in clinical situations
[5]. Phages multiply logarithmically in infected bacterial
cells, and the release of progeny phage occurs by lysis of
the infected cell at the end of the infection cycle, which
involves the holin-endolysin system [6,7]. Holins create a
lesion in the cytoplasmic membrane through which
endolysins gain access to the murein layer [7]. Endolysins
are peptidoglycan hydrolases that degrade the bacterial
cell wall, leading to cell lysis and release of progeny
© 2011 Paul et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
phages [8]. An undesirable side effect of this phenomenon from a therapeutic perspective is the development
of immunogenic reactions due to large uncontrolled
amounts of phages in circulation [9]. Such concerns
must be addressed before phage therapy can be widely
accepted [5,10].
This work features engineered bacteriophages that are
incapable of lysing bacterial cells because they lack endolysin enzymatic activity. We previously produced, as a
model, a recombinant lysis-deficient version of T4 bacteriophage that infects Escherichia coli [11,12]. Phages have
also been engineered to be non- replicating or to possess
additional desirable properties [13-15]. In an experimental
E. coli infection model, the improved survival rate of rats
treated with lysis-deficient T4LyD phage was attributed to
lower endotoxin release [16].
We wished to generate an endolysin-deficient phage
against a gram-positive bacterium, and chose S. aureus
because of its clinical relevance. S. aureus is a major pathogen responsible for a variety of diseases ranging from
minor skin infections to life-threatening conditions such as
sepsis. This pathogen is often resistant to all b-lactam antibiotics; vancomycin-resistant strains may become untreatable [17-19]. This organism is the most common cause of
nosocomial infections, and nasal carriage is implicated as a
risk factor [20]. In the United States alone, invasive methicillin-resistant S. aureus (MRSA) infections occur in
approximately 94,000 people each year, causing nearly
19,000 deaths [21]. Understandably, the progressive multidrug resistance of bacteria has motivated the re-evaluation
of phages as therapy for diverse bacterial infections [22].
We report here that the recombinant endolysin-deficient
S. aureus phage P954 kills cells without causing cell lysis
and forms plaques on a host that expresses a plasmidencoded heterologous endolysin, enabling its large-scale
production. The recombinant phage P954 was evaluated
for in vivo efficacy in an experimental mouse model and
found to protect mice from fatal S. aureus infection.
Page 2 of 9
containing pUC19 cloned into the HindIII site of S. aureus plasmid pC194 [24], was used as a source for the cat
gene. A shuttle vector containing the temperature-sensitive replication origin of S. aureus, pCL52.2, was used as
source for the replication origin [25]. The constitutive
Bacillus subtilis vegII promoter was derived from pRB474
[26]. All bacterial strains were cultured in liquid Luria
Bertani (LB) medium at 37°C on a rotary shaker (200
rpm) unless otherwise stated. Ampicillin, chloramphenicol, and tetracycline were used as needed. All chemicals
were obtained from Sigma-Aldrich, St. Louis, MO, USA
unless otherwise mentioned.
Propagation, concentration, and enumeration of
bacteriophages
Bacteriophage P954 is a temperate phage that was isolated
from the Ganges River (India) and amplified in S. aureus
strain RN4220. Briefly, S. aureus RN4220 was grown at 37°
C in LB medium to an absorbance of approximately 0.8 at
600 nm, infected with phage P954 at a multiplicity of infection (MOI) of 0.01, and cultured at 37°C until the culture
lysed completely. After centrifugation at 4100 × g for 10
min to remove cell debris, the bacteriophages were concentrated by centrifugation at 27,760 × g for 90 min. The bacteriophage titer was determined by enumerating plaqueforming units (PFUs) in serial 10-fold dilutions in LB medium and confirmed by the agar overlay method [27,28].
Preparation of phage P954 DNA and genome sequencing
Phage P954 DNA was prepared from a stock solution (1 ×
1012 PFU/ml). The concentrated phage preparation (1 ml)
was incubated at 37°C for 1 hr with DNase I (1 μg/ml) and
RNase A (100 μg/ml). The mixture was adjusted to contain 1% sodium dodecyl sulfate, 50 mM EDTA (pH 8.0),
and 0.5 μg proteinase K and incubated at 65°C for 60 min.
The mixture was then subjected to phenol-chloroformisoamyl alcohol (25:24:1) extraction, and the DNA was
precipitated [29]. Purified phage DNA was used for genome sequencing [GenBank: GQ398772].
Methods
Bacterial strains, plasmids, and growth conditions
E. coli strain DH5a [F80dlacZΔM15 Δ (lacZYA-argF)
recA1 endA1 hsdR17 supE44 thi-1 gyrA96relA1deoR] was
used as host for plasmid constructions and plasmid propagation. A restriction-deficient prophage-free S. aureus
strain RN4220 [23] was used for recombination, lysogenization, and phage enrichment. Clinical isolates of S. aureus were used to test phage sensitivity. A MRSA clinical
isolate (B911) was used in animal experiments to determine the in vivo efficacy of the endolysin-deficient phage
P954.
The plasmid pET21a (Novagen, USA) was used for
cloning and construction of endolysin disruption cassette.
The plasmid pSK236, an E. coli - S. aureus shuttle vector
Construction of plasmids for phage P954 endolysin
disruption
The phage P954 endolysin gene (753 bp) was amplified as
two separate fragments by polymerase chain reaction
(PCR). The first fragment (bp 1-376) was amplified with
forward primer 5’-CGGAATTCcatatgAAAACATACAGTGAAGCAAGAGCA-3’, containing an NdeI restriction site, and reverse primer 5’-CCGCCGCTgaattcTAAT
AAAGTGAGTACAGCC-3’, containing an EcoRI site.
The fragment was cloned into a pET21a vector at the
NdeI/EcoRI sites.
The second fragment (bp 377-753) was amplified with
forward primer 5’-CCGCCGGgaattcAGTATAAAAGTGAGGGCTTA-3’, containing an EcoRI site, and reverse
Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
primer 5’-CCaagcttTTAAAACACTTCTTTCACAATCAATCTCTC-3’, containing a HindIII site. The second
fragment was cloned in tandem with the first fragment,
thus generating the full-length phage P954 lysin gene
with an internal EcoRI site. The cat gene was isolated
along with its constitutive promoter from the S. aureus
- E. coli shuttle plasmid pSK236 by ClaI digestion.
Cohesive ends were filled with the Klenow fragment of
DNA polymerase I and ligated into the blunted EcoRI
site of the full-length phage P954 endolysin gene,
thereby disrupting it. The S. aureus-specific temperature-sensitive origin of replication from the shuttle vector pCL52.2 was introduced at the XhoI restriction site
of this construct to generate pGMB390.
Mitomycin C induction of phage P954 lysogens
The S. aureus RN4220 lysogen of phage P954 was inoculated in LB medium and incubated at 37°C with shaking
at 200 rpm for 16 hr. The cells were then subcultured in
LB medium at 2% inoculum and incubated at 37°C with
shaking at 200 rpm until the culture attained an absorbance of 1.0 at 600 nm. Mitomycin C was then added to
a final concentration of 1 μg/ml, and the culture was
incubated at 37°C with shaking at 200 rpm for 4 hr for
prophage induction.
Recombination and screening for recombinants
S. aureus RN4220 cells were transformed with pGMB390
by electroporation according to the protocol described by
Schenk and Laddaga [30] with a BioRad Gene Pulser, plated on LB agar containing chloramphenicol (10 μg/ml),
and incubated at 37°C for 16 hr. Chloramphenicol-resistant colonies were selected and grown in LB at 37°C until
the cultures reached an absorbance of 1.0 at 600 nm.
Recombination was then initiated by infecting these cells
with phage P954 (MOI = 3) for 30 min. Progeny phage
were harvested from the lysate as described previously,
lysogenized in S. aureus RN4220, and plated on LB agar
containing chloramphenicol (10 μg/ml) (round I).
Ninety-six chloramphenicol-resistant colonies were
picked up, grown, and induced with Mitomycin C. Cultures that did not lyse after the 16-hr Mitomycin C
induction were treated with 1% chloroform and lysed
with glass beads; the released phages were again lysogenized in S. aureus RN4220 (round II). Chloramphenicolresistant colonies of round II lysogens were similarly
grown and subjected to Mitomycin C induction. The
chloramphenicol-resistant lysogens that did not release
phages upon Mitomycin C induction were selected for
PCR analysis. Genomic DNA of the selected lysogens was
purified, and PCR was performed with different sets of
primers to confirm disruption of the phage P954 endolysin gene.
Page 3 of 9
Endolysin complementation for phage enrichment and
enumeration
The endolysin gene from a Podoviridae phage in our collection, P926, was cloned under the constitutive B. subtilis
vegII promoter in an E. coli - S. aureus shuttle vector constructed in our laboratory. This construct, designated
pGMB540, was used for trans-complementation of the
nonfunctional endolysin for propagation of the recombinant phage in lytic mode and for their enumeration. Plasmid pGMB540 was introduced into S. aureus strain
RN4220 by electroporation according to the protocol
described by Schenk and Laddaga [30]. Transformants
were selected on LB medium containing tetracycline
(5 μg/ml) and used as bacterial hosts for phage enrichment. Early log phase cells of S. aureus RN4220/
pGMB540 grown at 37°C were infected with the recombinant endolysin-deficient phage P954 (MOI = 0.1) and
incubated for an additional 3 to 4 hr until the culture
lysed. The phage-containing lysate was passed through a
0.2-μm filter, and the phages were enumerated on a lawn
of S. aureus RN4220/pGMB540 cells.
The endolysin-deficient phage P954 was also enriched
by induction. Briefly, the lysogen was grown at 37°C until
absorbance at 600 nm reached 1.0 and then induced with
1 μg/ml Mitomycin C at 37°C for 4 hr. The cells were pelleted and lysed by vortexing with glass beads. Cell debris
was removed by centrifugation at 5000 × g for 10 min,
and the phage-containing supernatant was passed through
a 0.2-μm filter.
Comparison of in vitro bactericidal activity of parent and
lysis-deficient phage P954
The parent and recombinant phages were compared for
host range and bactericidal activity. Ten MOI equivalent
of phage was added to 2 × 108 colony-forming units per
ml (CFU/ml) and incubated at 37°C for 90 min. Serial
10-fold dilutions of the mixture were plated on LB agar,
and residual viable cells (CFUs) were enumerated.
In vivo efficacy of endolysin-deficient phage P954 in
neutropenic mice
Animal experiments were performed at St. John’s Medical
College and Hospital, Bangalore, India. The experiments
were approved by the Institutional Animal Ethics Committee and the Committee for the Purpose of Control and
Supervision of Experiments on Animals (registration No.
90/1999/CPCSEA dated 28/4/1999).
Healthy male Swiss albino mice (6-8 weeks old, neutropenic) were used to evaluate in vivo efficacy. Neutropenia
was induced by intraperitoneal (IP) administration of
cyclophosphamide (100 mg/kg). In a preliminary study,
the lethality of a clinical MRSA isolate (B911) was determined in the mice (1 × 10 7 -1 × 10 8 CFU). We found
Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
that 5 × 107 CFU resulted in 80% mortality (LD80), and it
was therefore chosen as the challenge dose to evaluate
phage efficacy (data not shown).
In the efficacy experiment, mice were assigned to six
treatment groups (n = 8, each group). Four days after
cyclophosphamide treatment, the mice in groups 1-3
were challenged with B911 (200 μl, 5 × 10 7 CFU).
Groups 1 and 4 were then treated with 25 mM TrisHCl, pH 7.5 (negative control); groups 2 and 5 were
treated with two doses of endolysin-deficient phage
P954 prepared in 25 mM Tris-HCl, pH 7.5 at 200 MOI
equivalent (MOI relative to CFU at LD80); and groups 3
and 6 were treated with two doses of chloramphenicol
(50 mg/kg). The first treatment dose was administered
immediately after challenge; the second dose was administered 2 hr later. Mice were observed over 10 days for
occurrence of mortality. Survival analysis is plotted as
Kaplan-Meier survival curves using MedCalc statistical
software version 11.6.0.0 (Mariakerke, Belgium).
Results
Genome of phage P954
The 40761-bp phage P954 genome (Genome map provided as Additional file 1 Figure S1) is composed of linear
double-stranded DNA with a G+C content of 33.99%
[GenBank: GQ398772]. BlastN [31] searches with the
phage P954 nucleotide sequence showed it to be similar to
other sequenced staphylococcal phages in the NCBI database. The P954 genome matches that of S. aureus phage
phiNM3 (accession no. DQ530361) with pair-wise identity
of 66%. At least 69 open reading frames (ORFs) were predicted with the GeneMark program [32]. Bioinformatics
analysis revealed that 46 of the 69 ORFs are hypothetical/
conserved hypothetical proteins; the other 23 ORFs show
a high degree of homology to proteins from other staphylococcal phages in the database. The lysis cassette of this
phage was found to be similar to lysis systems of other
staphylococcal phages. The closest match to the phage
P954 holin gene was staphylococcal prophage phiPV8,
with 97% identity. The endolysin gene of phage P954 is
100% identical to the amidase gene from staphylococcal
phage phi13; the phage P954 integrase gene is 100% identical to ORF 007 of staphylococcal phage 85; and the
phage P954 repressor gene is 100% identical to the putative phage repressor of S. aureus subsp JH9. Our analysis
did not reveal the presence of any toxin encoding genes in
the phage P954 genome.
Screening of recombinants
The native phage endolysin gene was inactivated, and the
recombinant phage engendered by homologous recombination between phage P954 and plasmid pGMB390 in
S. aureus RN4220. Screening for S. aureus RN4220 lysogens harboring recombinant phage P954, in which
Page 4 of 9
endolysin was inactivated by insertion of the cat gene, was
carried out using chloramphenicol resistance as a marker.
Ninety-six colonies were obtained of which two lysogens
did not show lysis with Mitomycin C induction for up
to 16 hours. Phages mechanically released from these
colonies upon relysogenization yielded chloramphenicol
resistant lysogens that did not lyse upon Mitomycin C
induction. PCR analyses using two primer sets confirmed
disruption of the endolysin gene in all the recombinant
lysogens screened. Representative PCR profile of recombinant and parent phage lysogens is shown (Figure 1).
Mitomycin C induction of parent and endolysin-deficient
phage P954
We examined the prophage induction pattern and phage
progeny release from parent and endolysin-deficient phage
P954 lysogens. Absorbance and extracellular phage titers
were monitored every hour until the end of induction.
Induction of the parent phage P954 lysogen (B7) resulted
in cell lysis and gave a phage titer of 1 × 109 PFU/ml. In
contrast, the endolysin-deficient phage P954 lysogen did
not lyse and gave a phage titer of about 10 3 PFU/ml
(Figure 2).
Endolysin complementation for phage enrichment and
enumeration
Endolysin-deficient phage P954 could be enriched to
titers of up to 5 × 10 10 PFU/ml in S. aureus RN4220
that constitutively expressed phage P926 endolysin. This
strain was used also to determine titers of the endolysin-deficient phage preparations. When preparations of
the endolysin-deficient phage were spotted on a noncomplementing host, a zone of lysis characteristic of
“lysis from without” was observed at lower dilutions,
and no plaques were discernible (Figure 3a). The recombinant phage formed plaques only on the endolysincomplementing host (Figure 3b, c, d).
Comparison of in vitro bactericidal activity of parent and
lysis-deficient phage P954
Parent and recombinant endolysin-deficient phage P954
demonstrated comparable bactericidal activity when
tested on a panel of seven phage-sensitive and one
phage-resistant (B9030) clinical isolates, comprising
both methicillin-sensitive S. aureus (MSSA) and MRSA
strains, from our collection. Cell viability was reduced
by ≥ 90% with both phages (Figure 4). Similarly, the
host range of each phage was the same on a panel of 20
phage-sensitive and phage-resistant clinical isolates (data
provided as Additional file 2 Table S1).
In vivo efficacy of endolysin-deficient phage P954
An IP injection of the MRSA isolate B911 (5 × 107 cells/
mouse) resulted in the onset of disease in 80% of mice
Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
Page 5 of 9
a
b
c
d
Figure 1 Schematic and PCR analysis of parent and recombinant endolysin-deficient phage P954. Alignment of primers to the (a) parent
phage DNA template and (b) recombinant phage DNA template, in which the cat gene had been inserted into the endolysin gene (LYS). (c)
Endolysin forward and reverse primers yield a 750-bp PCR product of the parent phage P954 and 2400-bp product of the recombinant phage
P954. (d) The holin forward primer and endolysin reverse primer yield a 1000-bp PCR product with parent phage P954 and 2650-bp product of
the recombinant phage P954. Both PCR panels include lane 1: PCR buffer (negative control); lane 2: parent phage P954 lysogen B7, lane 3:
molecular weight marker (l/HindIII-EcoRI); lane 4: recombinant phage P954 lysogen H10.
(group 1), indicated by dullness, ruffled fur, and death
within 48 hr (Figure 5). However, IP administration of
endolysin-deficient phage P954 as two doses (immediately and after 2 hr) post B911 challenge fully protected
the mice against lethality (group 2). Similarly, chloramphenicol (dose regimen similar to phage) protected mice
against lethality (group 3); however, one animal died in
each of the chloramphenicol treatment groups of
unknown causes (groups 3 and 6). Endolysin-deficient
phage alone was not toxic or lethal to neutropenic mice,
demonstrating its safety (group 5). Endolysin-deficient
phage demonstrated significant efficacy against MRSA
B911in the tested animal model (P value = 0.0001).
Discussion
Bacteriophage endolysins are peptidoglycan hydrolases
that function at the end of the phage multiplication cycle,
lysing the bacterial cell and releasing new phages to
infect other bacteria. Many efforts to develop therapeutic
phages have focused on the lytic endpoint of phage infection to destroy the bacterium. However, cell lysis by
phage may present the problem of endotoxin release and
serious consequences as known in the case of antibiotics
[33]. Antibiotic-induced release of Lipotiechoic acids and
peptidoglycan (PG) in case of gram positive bacteria has
been shown to enhance systemic inflammatory responses
[34]. An endolysin-deficient phage does not degrade the
bacterial cell wall, thus progeny are not released until the
cell disintegrates or is lysed by other means. However,
the phage protein holin, produces an inner membrane
lesion at the end of the phage replication cycle, which
terminates respiration [7] and ensures killing of the cell.
In an in vivo situation, we can expect such dead cells to
be cleared rapidly by the host immune system.
Non-replicating genetically modified filamentous
phage which exerted high killing efficiency on cells with
minimal release of endotoxin is reported [13]. Higher
survival rate correlated with reduced inflammatory
response in case of infected mice treated with genetically modified phage [14]. A phage genetically engineered to produce an enzyme that degrades extracellular
polymeric substances and disperses biofilms is reported
[15].
Although temperate phages present the problem of lysogeny and the associated risk of transfer of virulence factors
through bacterial DNA transduction; we have used a temperate phage as a model for this study as the prophage status simplifies genetic manipulation. Because S. aureus
strains are known to harbor multiple prophages, which
could potentially interfere with recombination and engineering events, we elected to lysogenize phage P954 in a
prophage-free host, S. aureus RN4220. Our strategy was to
identify lysogens that harbored the recombinant endolysin-deficient phages, based on detection of phage P954
genes and the cat marker gene by PCR analysis (Figure 1).
In the recombination experiment, the 96 chloramphenicol resistant colonies obtained represented recombinant endolysin-inactivated prophage some of which lysed
upon Mitomycin C induction. We suspected that the parent phage could also have lysogenized along with the
Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
Page 6 of 9
Figure 2 Mitomycin C induction of parent and endolysin-deficient phage P954 lysogens. (a) Growth profiles of the parent (B7) and
endolysin-deficient (H10) phage P954 lysogens after Mitomycin C induction showing absorbance of cultures at 600 nm. The graph is
representative of two experiments. The error bars represent mean plus standard deviation (n = 3) (b) Phage release into the culture medium
from parent (B7) and endolysin-deficient (H10) phage P954 lysogens after Mitomycin C induction. The graph is representative of 2 experiments.
recombinant phage. We overcame the problem by
repeating the induction of chloramphenicol resistant
lysogens and lysogenization of the phages produced.
When we assessed the prophage induction pattern and
phage progeny release of parent and endolysin-deficient
phage P954 lysogens, we found that the absorbance of
the culture remained unaltered and the extracellular
phage titer was minimal with the recombinant phage
lysogen. We observed a low phage titer 3 to 4 hours
after induction, presumably due to natural disintegration
Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
Page 7 of 9
Figure 3 Complementation with heterologous endolysin gene for enrichment of endolysin-deficient phage P954. Ten-fold serial
dilutions of endolysin-deficient phage P954 (5 × 1010 PFU/ml) spotted on (a) S. aureus RN4220 lawn and (b) complementing host pGMB540/S.
aureus RN4220, which expresses a heterologous endolysin. Plaque assay of enriched endolysin-deficient phage P954 on (c) non-complementing
host S. aureus RN4220 and (d) complementing host pGMB540/S. aureus RN4220.
and lysis of a small percentage of the cell population. In
contrast, we observed lysis of the culture by the parent
phage with increasing phage titer in the lysate, as
expected (Figure 2).
Complementation of the lysis-deficient phenotype was
achieved using a heterologous phage P926 from our collection. Supplying the endolysin gene in trans allowed the
recombinant phage to form plaques (Figure 3b, d). This
Figure 4 Bactericidal activity of parent and lysis-deficient phage P954. Bactericidal activity of parent and lysis-deficient phage P954 (10 MOI
equivalent) on eight clinical isolates of MRSA (B910, B954, B9053, B9194, B9195) and MSSA (B911, B9007, B9030). Phage resistant isolate indicated
with asterix (*). The error bars represent standard deviation (n = 3, single experiment).
Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
Page 8 of 9
Figure 5 In vivo efficacy of endolysin-deficient phage P954. Survival of mice challenged with clinical MRSA isolate (B911). Groups 1-3 were
challenged with MRSA (5 × 107 cells per mouse). Groups 4-6 were not challenged with MRSA and served as controls. The following treatments
were administered: groups 1 and 4 (25 mM Tris-HCl, pH 7.5); groups 2 and 5 (two doses of endolysin-deficient phage P954, 200 MOI); groups 3
and 6 (two doses of chloramphenicol, 50 mg/kg).
was used to determine titers of the endolysin-deficient
phage throughout our study, and provided an excellent
method for efficient phage enrichment. Use of a heterologous phage endolysin enabled the recombinant phage to
exhibit the lysis-deficient phenotype even after several
rounds of multiplication. In vitro activity of the endolysindeficient phage against MSSA and MRSA was comparable
to that of the parent phage (Figure 4). Further, the recombinant phage was able to rescue mice from fatal MRSA
infection (Figure 5), similar to the parent phage (data not
shown). Future studies will have to compare systemic
responses and outcomes of treatment with native and
endolysin- deficient phage in S. aureus infection.
This work demonstrates the potential of disrupting the
endolysin gene to reduce the number of phages that are
otherwise released post-infection by their lytic parent
phage. In clinical situations, this would provide the
advantage of a defined dosage, which is an important
concern raised against phage therapy [5,35], as well as
lower immune response and reduced endotoxin release
when using gram-negative bacteria. This is the first
report of a gram-positive endolysin-deficient phage. Our
results demonstrate the therapeutic potential of engineered phages in clinical applications.
Conclusions
We developed a modified bacteriophage against S. aureus by insertional inactivation of its endolysin gene,
which renders it incapable of host cell lysis. This phage
is lethal to cells it infects, with little or no release of
progeny phage. We showed that the disrupted endolysin
could be complemented with a functional heterologous
endolysin gene to produce this phage in high titers. To
our knowledge, this is the first report of a gram-positive
endolysin-deficient phage. Further, we demonstrate its
therapeutic potential in an experimental infection model
in mice, in which the lysis-deficient phage P954 protects
against lethal MRSA.
Additional material
Additional file 1: Figure S1 - Genome map of phage P954. Phage
P954 genome is similar in organization to other known temperate
staphylococcal phages. The organization of the genome is modular, with
genes involved in lysogeny, replication, DNA packaging, tail assembly,
and lysis arranged sequentially).
Additional file 2: Table S1 - Comparison of host range of parent
and endolysin deficient phage P954. The host range of both the
phage were same on a panel of 20 phage-sensitive and phage-resistant
isolates.
Paul et al. BMC Microbiology 2011, 11:195
http://www.biomedcentral.com/1471-2180/11/195
List of Abbreviations
cat: Chloramphenicol acetyltransferase; CFU: Colony-forming unit; IP:
Intraperitoneal; LB: Luria Bertani; LD80: Lethal dose that results in 80% mortality;
MOI: Multiplicity of infection; MRSA: Methicillin-resistant Staphylococcus aureus;
MSSA: Methicillin-sensitive Staphylococcus aureus; PFU: Plaque-forming unit
Acknowledgements
S. aureus RN4220 was a kind gift from Dr. Richard Novick, Skirball Institute,
New York. The plasmid pRB474 was kindly provided by Prof. Ry Young,
Texas A&M University, Texas. Plasmids pCl52.2 and pSK236 were kindly
provided by Prof. Ambrose Cheung, Dartmouth Medical School, Hanover.
The authors would like to thank D. Murali, E. Bhavani, A. R. Thaslim Arif of
Gangagen Biotechnologies, and Dr. Sudha Suresh, Pharmacology Division of
St. John’s Medical College and Hospital, Bangalore, for assistance with animal
experiments. The authors wish to thank Dr. M. Jayasheela and Dr. Anand
Kumar for reviewing the manuscript.
Author details
Gangagen Biotechnologies Pvt Ltd, No. 12, 5th Cross, Raghavendra Layout,
Tumkur Road, Yeshwantpur, Bangalore-560 022, India. 2Department of
Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto,
ON M5S 1A8, Canada. 3Department of Animal Husbandry, Veterinary
Dispensary, Yediyur, Kunigal Taluk, Tumkur- 572142, India. 4Lupin Limited,
Biotechnology R & D, Gat #1156, Ghotawade Village, Mulshi Taluka, Pune411042, India.
Page 9 of 9
12.
13.
14.
15.
16.
17.
18.
1
Authors’ contributions
All the authors were affiliated with Gangagen Biotechnologies Pvt. Ltd.
when this work was carried out. JR conceived of the concept underlying this
study and contributed to the discussion in the manuscript. SP and BS
participated in study design and coordination and contributed to data
interpretation. VDP, SSR, and SS carried out cloning and generation of the
recombinant phage. SH and NK performed in vivo studies. VDP and SSR
helped draft the manuscript. All authors read and approved the final
manuscript.
Competing interests
Authors SP, BS, and JR are inventors on an issued patent (US Patent No
6,896,882) describing the concept of lysin-deficient bacteriophages as
therapeutic agents for the control of bacterial infection and methods of
developing such phages. Authors have assigned rights to Gangagen Inc.,
which is a current employer of JR, BS, SSR, SS, and SH and a previous
employer of SP, VDP, and NK.
Received: 6 April 2011 Accepted: 31 August 2011
Published: 31 August 2011
References
1. Barrow PA, Soothill JS: Bacteriophage therapy and prophylaxis:
rediscovery and renewed assessment of potential. Trends Microbiol 1997,
5:268-271.
2. Thacker PD: Set a microbe to kill a microbe: Drug resistance renews
interest in phage therapy. JAMA 2003, 290:3183-3185.
3. Soothill JS, Hawkins C, Anggard EA, Harper DR: Therapeutic use of
bacteriophages. Lancet Infectious Diseases 2004, 4:544-545.
4. Lang L: FDA approves use of bacteriophages to be added to meat and
poultry products. Gastroenterology 2006, 131:1370-1372.
5. William Summers C: Bacteriophage therapy. Annu Rev Microbiol 2001,
55:437-451.
6. Young R: Bacteriophage lysis: mechanism and regulation. Microbiol Rev
1992, 56:430-81.
7. Young RJ: Bacteriophage holins: deadly diversity. Mol Microbiol Biotechnol
2002, 4:21-36.
8. Loessner MJ: Bacteriophage endolysins - current state of research and
applications. Current Opinion in Microbiology 2005, 8:480-487.
9. Merril CR, Biswas B, Carlton R, Jensen NC, Creed GJ, Zullo S, Adhya S: Longcirculating bacteriophage as antibacterial agents. Proc Natl Acad Sci 1996,
93:3188-3192.
10. Projan S: Phage-inspired antibiotics? Nat Biotechnol 2004, 22:185-91.
11. Padmanabhan S, Sriram B, Sagar P, Shashikala V, Ramachandran J:
Insertional inactivation of the T4 lysozyme gene: Model for absolute
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
lysis-defectives in phage therapy. ASM Conference on the New Phage
Biology: the ‘Phage Summit’:1-5 Aug 2004; Key Biscayne, Florida, USA .
Ramachandran J, Sriram P, Sriram B: Lysin deficient bacteriophages having
reduced immunogenecity., US Patent No; 6,896,882.
Hagens S, Bläsi U: Genetically modified filamentous phage as bactericidal
agents: a pilot study. Lett Appl Microbiol 2003, 37:318-323.
Hagens S, Habel A, von Ahsen U, von Gabain A, Bläsi U: Therapy of
experimental pseudomonas infections with a nonreplicating genetically
modified phage. Antimicrob Agents Chemother 2004, 48:3817-3822.
Lu TK, Collins JJ: Dispersing biofilms with engineered enzymatic
bacteriophage. Proc Natl Acad Sci 2007, 104:11197-11202.
Matsuda T, Freeman TA, Hilbert DW, Duff M, Fuortes M, Stapleton PP,
Daly JM: Lysis-deficient bacteriophage therapy decreases endotoxin and
inflammatory mediator release and improves survival in a murine
peritonitis model. Surgery 2005, 137:639-646.
Hiramatsu K, Katayama Y, Yuzawa H, Ito T: Molecular genetics of methicillinresistant Staphylococcus aureus. Int J Med Microbiol 2002, 292:67-74.
Smith TL, Pearson ML, Wilcox KR, Cruz C, Lancaster MV, Robinson-Dunn B,
Tenover FC, Zervos MJ, Band JD, White E, Jarvis WR: Emergence of
vancomycin resistance in Staphylococcus aureus. GlycopeptideIntermediate Staphylococcus aureus Working Group. N Engl J Med 1999,
340:493-501.
CDC: Staphylococcus aureus Resistant to Vancomycin - United States
2002. MMWR 2002, 51:565-567.
Perl TM, Golub JE: New approaches to reduce Staphylococcus aureus
nosocomial infection rates: treating S. aureus nasal carriage. Ann
Pharmacother 1998, 32:S7-16.
Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, Harrison LH,
Lynfield R, Dumyati G, Townes JM, Craig AS, Zell ER, Fosheim GE,
McDougal LK, Carey RB, Fridkin SK: Invasive methicillin-resistant
Staphylococcus aureus infections in the United States. JAMA 2007,
298:1763-1771.
Merril CR, Scholl D, Adhya SL: The prospect for bacteriophage therapy in
Western medicine. Nat Rev Drug Discov 2003, 2:489-497.
Kreiswirth BN, Löfdahl S, Betley MJ, O’Reilly M, Schlievert PM, Bergdoll MS,
Novick RP: The toxic shock syndrome exotoxin structural gene is not
detectably transmitted by a prophage. Nature 1983, 305:709-712.
Mahmood R, Khan SA: Role of upstream sequences in the expression of
the Staphylococcal enterotoxin B gene. J Biol Chem 1990, 265:4652-4656.
Lee CY: Cloning of genes affecting capsule expression in Staphylococcus
aureus strain M. Mol Microbiol 1992, 6:1515-1522.
Jankovic I, Egeter O, Brückner R: Analysis of catabolite control protein Adependent repression in Staphylococcus xylosus by a genomic reporter
gene system. J Bacteriol 2001, 183:580-586.
Adams MH: Bacteriophages New York: Interscience Publishers; 1959.
Carlson K: Working With Bacteriophages: Common Techniques And
Methodological Approaches. In Bacteriophages: Biology and Applications.
Edited by: Kutter E, Sulakvelidze A. CRC press; 2005:437-490.
Sambrook J, Russel DW: Molecular Cloning: A Laboratory Manual. 3 edition.
Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York; 2001.
Schenk S, Laddaga RA: Improved method for electroporation of
Staphylococcus aureus. FEMS Microbiol Lett 1992, 73:133-138.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment
search tool. J Mol Biol 1990, 215:403-410.
Lukashin A, Borodovsky M: GeneMark.hmm: new solutions for gene
finding. Nucleic Acids Research 1998, 26:1107-1115.
Shenep JL, Barton RP, Mogan KA: Role of antibiotic class in the rate of
liberation of endotoxin during therapy for experimantal gram-negative
bacterial sepsis. J Infect Dis 1985, 151:1012-1018.
Van Langevelde P, Ravensbergen E, Grashoff P, Beekhuizen H,
Groeneveld PH, Van Dissel JT: Antibiotic-induced cell wall fragments of
Staphylococcus aureus increase endothelial chemokine secretion and
adhesiveness for granulocytes. Antimicrob Agents Chemother 1999,
43:2984-2989.
Schoolnik GK, Summers WC, Watson JD: Phage offer a real alternative.
Nature Biotechnol 2004, 22:505-506.
doi:10.1186/1471-2180-11-195
Cite this article as: Paul et al.: Lysis-deficient phages as novel
therapeutic agents for controlling bacterial infection. BMC Microbiology
2011 11:195.